Device for the study of living cells

ABSTRACT

A cell study device, comprising, a base layer, a planar conduit defining layer, including a conduit cut out of the layer; and a planar cover layer which defines a capillary flow channel in said conduit layer, said conduit layer and said cover layer acting as side walls for said capillary flow channel, wherein said layers are formed of materials that do not interfere with cell behavior over a period of at least 5 hours when loaded with aqueous solution.

RELATED APPLICATIONS

This application is a National Phase of PCT Patent Application No.PCT/IL2008/001678 having International filing date of Dec. 25, 2008,which is a continuation-in-part (CIP) of PCT Patent Application No.PCT/IL2008/001492 filed Nov. 13, 2008, which is a continuation-in-part(CIP) of U.S. patent application Ser. No. 11/940,996 filed Nov. 15,2007.

PCT Patent Application Nos. PCT/IL2008/001678 and PCT/IL2008/001492 bothclaim the benefit of priority under 35 USC 119(e) of U.S. ProvisionalPatent Application No. 61/006,130 filed Dec. 26, 2007.

This application is also a continuation-in-part (CIP) of U.S. patentapplication Ser. No. 12/712,232 filed Feb. 25, 2010, which claims thebenefit of priority under 35 USC 119(e) of U.S. Provisional PatentApplication No. 61/155,186 filed Feb. 25, 2009.

U.S. patent application Ser. No. 12/712,232 is also acontinuation-in-part (CIP) of PCT Patent Application No.PCT/IL2008/001678.

The contents of the above applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

The present invention, in some embodiments thereof, relates to the fieldof devices for biology, and more particularly, but not exclusively, todevices useful for the study and/or maintenance of a plurality livingcells.

BACKGROUND OF THE INVENTION

The study of cell behavior is important in many fields includingbiology, medicine and pharmacology. Since cell-functions include manyinterrelated pathways, cycles and chemical reactions and since there isa large variation of cell biochemistry amongst similar cells, the studyof a bulk of cells, whether the bulk is homogenous or heterogeneous,does not usually provide sufficiently detailed or interpretable results:rather a comprehensive study of cell biological activity is oftenadvantageously performed by examining single isolated living cells asindividuals. The use of single-cell assays is an important tool forunderstanding biological systems and the influence thereupon of variousstimuli such as exposure to active entities.

In order to understand cell behavior, for example, such as the responseto stimuli such as various biological modulators, two fundamentalresearch capabilities are often desirable (i) the ability to tracktemporal behavior of large groups of cells as individuals for periods ofminutes, hours and even days and (ii) the ability to identify and studycell heterogenity, a phenomenon existing even in synchronized celllines.

SUMMARY OF THE INVENTION

Some embodiments of the present invention provide a device including anarray of wells and capillary channels to lead fluid form and to thecells, optionally formed on a glass slide and being useful the study ofa plurality of cells as individuals. In an exemplary embodiment of theinvention, the device is constructed of materials and has a structureselected so that the device has a desired degree of capillary flow. Inan exemplary embodiment of the invention, the device is formed ofmultiple layers with apertures therein, laid one on the other anddefining capillary flow in spaces between.

There is provided in accordance with an exemplary embodiment of theinvention, a cell study device, comprising:

a base layer;

a planar conduit defining layer, including a conduit cut out of thelayer; and

a planar cover layer which defines a capillary flow channel in saidconduit layer, said conduit layer and said cover layer acting as sidewalls for said capillary flow channel,

wherein said layers are formed of materials that do not interfere withcell behavior over a period of at least 5 hours when loaded with aqueoussolution.

In an exemplary embodiment of the invention, the device comprises a cellholding area defined in fluid contact with said capillary flow channel.Optionally, said cell holding area includes at least one orientationmark visible under microscopy. Optionally or alternatively, said cellholding area is masked by a masking layer underlying said conduit layer.

In an exemplary embodiment of the invention, said cell holding area ismounted on said base layer.

In an exemplary embodiment of the invention, said cover layer includesan air hole for air release form said capillary flow channel.

In an exemplary embodiment of the invention, said capillary flow channeldefines a substantially sealed waste reservoir with no fluid exit.

In an exemplary embodiment of the invention, said capillary flow channeldefines a substantially sealed waste reservoir with an absorbentmaterial as a fluid exit.

In an exemplary embodiment of the invention, said cover defines a fluidinlet area for said capillary flow conduit.

In an exemplary embodiment of the invention, said device is packaged invacuum.

In an exemplary embodiment of the invention, said conduit layer ispermanently adhesive to said cover layer. Optionally, said device isprovided with a removable non-stick layer intermediate most of saidcover layer and said conduit layer.

In an exemplary embodiment of the invention, said conduit layer istemporarily adhesive to said cover layer.

In an exemplary embodiment of the invention, said device is formedessentially of layered planar layers.

In an exemplary embodiment of the invention, said conduit layer isadhesive on both its faces.

In an exemplary embodiment of the invention, said layers are selected ofdissimilar materials with dissimilar contact angles with fluids.

In an exemplary embodiment of the invention, said device has the formfactor of a standard microscope slide.

In an exemplary embodiment of the invention, said device has the formfactor of a standard microtitter plate.

In an exemplary embodiment of the invention, said materials do notinterfere for at least 24 hours.

In an exemplary embodiment of the invention, said cover layer isopenable for access to said capillary conduit and removal of cellstherefrom.

There is provided in accordance with an exemplary embodiment of theinvention, a kit, comprising:

(a) a cell study device including a capillary flow conduit and a cellholding area; and

(b) at least an indication of one or both of a capillary flow rate and acell dislocation rate therein. Optionally, the kit comprises a pluralityof different cell study devices, each with different sets of flow rateand dislocation rate. Optionally or alternatively, said indication isper one or both of fluid property and cell type. Optionally oralternatively, said indication comprises a machine input indication.Optionally or alternatively, said indication comprises a human readableindication.

In an exemplary embodiment of the invention, the kit comprisesinstructions explaining said indications.

In an exemplary embodiment of the invention, the kit comprises softwareon a computer readable media for using said indications.

There is provided in accordance with an exemplary embodiment of theinvention, a pre-assembled and packaged cell study device including:

a capillary flow conduit;

a cell holding area; and

at least one non-adhesive layer, designed for removal and interferingwith adhesion of at least two parts of said device, said interferinginactivating said capillary flow conduit. Optionally, said adhesion ispermanent, when said non-adhesive interfering layer is removed.

There is provided in accordance with an exemplary embodiment of theinvention, a method of assembling a cell study device, comprising:

(a) selecting desired device characteristics;

(b) selecting device components from a set of pre-manufacturedcomponents, said selected components selected to interact to providesaid characteristics; and

(c) assembling said components to provide said device with said desiredcharacteristics.

In an exemplary embodiment of the invention, said set includescomponents of difference wettability. Optionally or alternatively, saidset includes components defining different capillary flow conduitgeometries. Optionally or alternatively, said set includes componentsdefining different cell holding area geometries.

There is provided in accordance with an exemplary embodiment of theinvention, a cell study device, comprising:

a capillary flow conduit having an inlet, enclosed on four sides andsealed on a distal end; and

at least one air release aperture defined in a top of said conduit.

There is provided in accordance with an exemplary embodiment of theinvention, a cell study device, comprising:

at least one array of cell holders;

a double sided adhesive layer masking some of said cell holders; and

a layer defining one or both of capillary channels and walls mounted onsaid adhesive layer. Optionally, said device comprises a plurality offluidicly disconnected cell holder arrays. Optionally or alternatively,said adhesive layer is apertured. Optionally or alternatively, saidwalls are at least 2 mm high.

There is provided in accordance with an exemplary embodiment of theinvention, a method of forming a cell study device, comprising adheringa plurality of precut dry or wet layers by applying pressure anddefining at least one capillary flow channel between layers, thereby.Optionally, the method comprises:

annealing said device under heat;

soaking said device in a solvent matched to said adhering; and

washing away said solvent.

In an exemplary embodiment of the invention, the method comprisesembossing a cell holding area on said device.

In an exemplary embodiment of the invention, the method comprisesessentially of said adhering

In an exemplary embodiment of the invention, said adhering comprisesadhering, or post treatment, in a low atmospheric pressure condition.

There is provide din accordance with an exemplary embodiment of theinvention a method of studying cells, comprising:

(i) determining one or both of desired flow rates for fluid used duringa study and a rate of cell dislocations for a cell type or aggregateused during the study;

(ii) selecting a cell study device including a capillary flow conduitand a cell holding area to match said determinations; and

(iii) using said selected device with said cell type in said study.

In an exemplary embodiment of the invention, selecting comprisesselecting from a plurality of devices in a kit.

In an exemplary embodiment of the invention, selecting comprisesselecting according to a catalog.

In an exemplary embodiment of the invention, selecting comprisesrecommending by a computer.

In an exemplary embodiment of the invention, selecting comprises adevice according to a contact angle between fluid used in the study andsaid capillary flow conduit.

In an exemplary embodiment of the invention, selecting comprises adevice according to a conduit cross-section.

In an exemplary embodiment of the invention, said device has a capillaryflow rate of less than 1 micro-liter per second.

In an exemplary embodiment of the invention, said device has a celldislocation rate of less than 10% per said study.

In an exemplary embodiment of the invention, said device includes atleast one baffle to control capillary flow rate.

In an exemplary embodiment of the invention, said device includes asubstantially sealed exit reservoir with at least one air hole tocontrol capillary flow rate.

In an exemplary embodiment of the invention, said device includes sidewalls in said capillary flow conduit which control capillary flow rate.

In an exemplary embodiment of the invention, said device includes one ormore changes in geometry at said cell holding area, which changescontrol cell dislocation rate.

In an exemplary embodiment of the invention, said device allowsapplication of cells directly to said cell holding area withoutcapillary flow.

In an exemplary embodiment of the invention, said selecting comprisesselecting according to a desired delivery rate of cells along saidcapillary flow to said cell holding area.

In an exemplary embodiment of the invention, said cell holding areacomprises non-adhesive picowells.

In an exemplary embodiment of the invention, the method comprisesmodifying a fluid used during a study to maintain said desired rate.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE FIGURES

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying Figures. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of some embodiments of the invention. In this regard, thedescription taken with the Figures makes apparent to those skilled inthe art how some embodiments of the invention may be practiced.

In the Figures:

FIG. 1A is a schematic depiction of a device useful for the study ofcells, disassembled to show components thereof, in accordance with anexemplary embodiment of the invention;

FIG. 1B is a reproduction of a SEM image of a well array of a device forthe study of cells, in accordance with an exemplary embodiment of theinvention;

FIGS. 2 and 3 are schematic depictions of a device for the study ofcells, in accordance with an exemplary embodiment of the invention;

FIGS. 4A-4F depict the use of a device such as depicted in FIGS. 1, 2and 3 for the study of cells, in accordance with an exemplary embodimentof the invention;

FIGS. 5A-5G schematically depict various devices useful for the study ofcells, in accordance with exemplary embodiments of the invention;

FIGS. 6A and 6B illustrate an alternative cell study device, inaccordance with an exemplary embodiment of the invention;

FIGS. 6C-6H illustrate exemplary sizes for parts of the device of FIGS.6A-6B;

FIG. 7 illustrates an alternative design of a device for studying cells,in accordance with an exemplary embodiment of the invention;

FIG. 8 is a flowchart of a method of using the device of FIGS. 6A-6H, inaccordance with an exemplary embodiment of the invention;

FIGS. 9A and 9B illustrate devices manufactured using double-sided tape,in accordance with exemplary embodiments of the invention;

FIG. 10A illustrates a dual-array slide design, in accordance with anexemplary embodiment of the invention; and

FIGS. 10B-10D illustrate additional multi-array slides, in accordancewith exemplary embodiments of the invention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to the studyof living cells and, more particularly, but not exclusively, to a deviceincluding an array of wells, the device useful the study of a pluralityof cells as individuals. In an exemplary embodiment of the invention,what is provided is means and methods and methods of manufacture, of asupport system including capillary flow to and from a cell study area.

In an exemplary embodiment of the invention, what is provided is amicroscope slide including a picowell array and using capillary actionto provide fluid to and from the well array. Optionally, the rate offlow is controlled by design methods and/or by user selection.Optionally, the device is pre-assembled. Optionally or alternatively,the device is formed of a plurality of planar layers with aperturesformed therein. Optionally, one of the apertures serves to mask anunderlying cell holding array. Optionally or alternatively, theapertures define one or more capillary flow channels. Optionally oralternatively, the apertures define an air release opening.

In an exemplary embodiment of the invention, the device is designed tobe a single piece device with a cover which can seal to underlyinglayers and define capillary conduits therewith. Optionally, a separatinglayer is provided between the cover and underlying layers, at least overpart of their interface, and is selectively removed by a user, duringuse.

In an exemplary embodiment of the invention, at least some of the layersare adhesive layers.

In an exemplary embodiment of the invention, what is provided is ahigh-content single live cell assay device which allows the study ofreal-time responses to stimuli in large and heterogeneous cellpopulations at an individual cell level. Such a device may be used toone or more of:

study each cell from a plurality of cells as an individual, observingthe real-time response of the cells to intervention using probes tomonitor morphological responses, intracellular and extra-cellularparameters, as well as cell-cell interaction;

perform long-term, non-intrusive, repeated measurements on intact,living, adherent or non-adherent cells, including bone marrow cells;

perform multiple functional assays on living cells, followed byimmuno-staining and chromatic staining on the same cells followingfixation;

perform kinetic measurements of non-synchronous activities in individualcells;

analyze and compare actual quantitative measurements of sub-populationsand individual cells as opposed to recording the mean values of entirepopulation; and/or

perform quantitative measurements on a cellular-to-molecular level, forexample, assays, killing cells in situ and then bursting them in situfor further analysis, feeding cells various substrates and/or exposingindividual or groups of cells to various stimulus. In some embodiments,the cell holding sections include both individuals cells and groups ofcells, for example, in different parts of the cell holding area.

Some devices and methods for multiple single cell study are taught inthe PCT Patent Applications published as WO2003/035824, WO2004/113492,WO2005/007796, WO2006/003664, WO2006/080000 and WO2007/052245 whichinclude Inventor Mordechai Deutsch, all of which are included byreference as if fully set forth herein. Features of such devices may beused in accordance with some exemplary embodiments of the invention.

“For example, WO2006/080000 describes “It is important to note that someembodiments of the present invention are related to embodiments ofunpublished copending PCT Patent Application No. IL04/00571 of theApplicant filed 27 Jun. 2004. In PCT Patent Application No. IL04/00571are taught picowell-bearing carriers having a variety of innovativefeatures. One aspect of the teachings of PCT Patent Application No.IL04/00571 is of picowells configured to influence cell proliferation ofcells held therein. In one embodiment, carriers having picowells of achangeable size is taught. In another embodiment, carriers configured todelay proliferation of cells held therein, for example by delaying orpreventing cell adhesion, are taught. In another embodiment, carriersconfigured so as to allow cells to grow into or through the carrier aretaught. The above-described embodiments are preferably implemented bymaking the picowells of or coating the picowells with a material withthe desired properties. In some embodiments, the inner surface of apicowell with which a held cell makes contact is configured to have thedesired property, influence or effect. Preferred materials from which tomake carriers listed in PCT Patent Application No. IL04/00571 includepolydimethylsiloxane, elastomers (such as silicon rubber), polymerizedpara-xylylene molecules, polymerized derivatives of para-xylylenemolecules and gels (especially hydrogels). In some embodiments, theinner surface of a picowell with which a held cell makes contact isconfigured to have the desired property, influence or effect.

An additional aspect of PCT Patent Application No. IL04/00571 is theteaching of a gel cover for picowell-bearing components. The gel coveris configured to prevent cells held in a picowell from exiting thepicowell due to jostling, incidental fluid flows or during movement ofthe carrier.

The advantages of a picowell-bearing carrier made of a gel, of apicowell gel-cover or a gel carrier covered with a gel cover include,depending on the embodiment, that active entities may be integrated intothe gel, that active entities may be contacted with the cell bydiffusion through the gel, that diffusion of released compounds isslowed down allowing identification of which cell released a givencompound, that proliferation of cells held therein is delayed but oncecells begin to proliferate, that allows proliferation into and throughthe gel matrix”.

In an exemplary embodiment of the invention, devices for the study ofcells include a picowell-bearing component. In some embodiments, apicowell-bearing component is a component having at least one, butgenerally a plurality of picowells, each picowell configured to hold atleast one cell. The term “picowell” is general and refers to a “smallwell”, that is physical feature that localizes a cell (or group ofcells) to a specific area on a planar surface of the picowell-bearingcomponent using by physical confinement. In some embodiments, apicowell-bearing component is a “carrier”, a substantially planarcomponent such as a chip, plate, sheet or slide. The term “picowell” isgenerally a discrete cavity of a size and shape suitable for retainingcells therein where the size and shape are defined by some physicalfeatures such as walls. The term “picowell” includes physical featuressuch as wells, dimples, depressions, pits, tubes and enclosures.

Since cells (or cell spheroids) typically range in size from about 1micrometers to about 400 (e.g., oocytes) micrometers diameter there ismay not be a single picowell size that is appropriate for holding asingle cell of any type. That said, the dimensions of the typicalindividual picowell in the picowell-bearing components optionally havedimensions suitable for accommodating cells having diameters of betweenabout 1 micrometer up to about 500 micrometers, depending on the exactimplementation, for example round or hexagonal picowells having adiameter of from about 1 micrometer up to about 500 micrometers. Forexample, a picowell-bearing component of a device designed for the studyof single isolated 8 to 20 micrometer diameter cells typically haspicowells of dimensions of about 20 micrometers. In some embodiments,larger picowells are used to study the interactions of a few cells heldtogether in one picowell. For example, a 200 micrometer picowell isrecognized as being useful for the study of cell spheroids or theinteractions of two, three or more cells, for example, as discussed inthe PCT Patent Application published as WO 2003/035824.

A feature that increases the utility of some embodiments of thepicowell-bearing devices is that the picowells are juxtaposed, that isto say the walls separating the individual picowells are thin (relativeto the size of the picowells) so that the area occupied by apicowell-array is substantially entirely made up of picowells withlittle or no inter picowell area. In an exemplary embodiment of theinvention, walls separating picowells are less than 1 micrometer wide sothat the inter-picowell area of the picowell array makes up only a minorpercentage of the total area of the picowell-array. This feature canallow near tissue-density packing of cells, especially in embodimentsconfigured to hold a single cell in each picowell.

In an exemplary embodiment of the invention, at least some of thepicowells are not etched or embossed and therefore are full and areoptionally used for optical alignment and/or as landmarks.

In an exemplary embodiment of the invention, a 2.2 mm by 2.2 mmpicowell-array of hexagonally-packed juxtaposed picowells having a 10micrometer diameter includes about 61,600 picowells. Optionally oralternatively, this feature allows simple loading of the picowells withcells: a liquid containing suspended cells is introduced in the volumeabove picowells of the appropriate size. Since there is littleinter-picowell area, cells, when they settle, mostly settle in thepicowells. After the cells settle, a flow of liquid applied in parallelto the surface of the picowell array either washes away cells resting ona wall separating picowells or leaning on another cell in a picowell, orpushes such a cell into an unoccupied picowell. In such a way, the onlycells in the proximity of the picowell array are cells held in apicowell, where each picowell holds only the number of cells for whichit is configured, for example, one cell per picowell, two cells perpicowell or three cells per picowell.

In an exemplary embodiment of the invention, the picowell-bearingcomponent is a thin, transparent carrier and the cells are observed frombelow with an inverted microscope or similar device, for example bydetection of light emitted by fluorescence or direct optical observationof the cells. In such embodiments, it is important that the bottoms ofthe picowells on which the cells rest be as coplanar as possible:coplanarity allows for optical observation of many cells (whether byscanning or simultaneously using a wide-angle observation component)without the need for time consuming and difficult-to-implementrefocusing. In such embodiments it is also important that the carrier beas flat and planar as possible, be of a uniform thickness and be ashomogenous as possible so that the carrier be as optically neutral aspossible, to allow observation of the cells with as little distortion aspossible.

In an exemplary embodiment of the invention, imaging is form above, forexample, if the device is configured as a microscope slide, for example,in size, shape and transparency, or in another standard form factor,such as a microtitter plate or any other common lab substrate.

In an exemplary embodiment of the invention, the use of a device forstudying cells including a picowell-bearing component typically involvesloading picowells with cells in a physiological medium and thenobserving the cells as individuals while exposing the cells to variousstimuli to study the effect of the stimuli on the individual cells.

In some instances, it is desirable to expose cells to a first stimulus,for example to a first medium containing a first active agent and aftera time to expose the cells to a second stimulus, for example to a secondmedium containing a second active agent. In an exemplary embodiment ofthe invention, the first medium is preferably entirely replaced with thesecond medium in proximity with the cells held in the picowell array.

In an exemplary embodiment of the invention, a device is provided wherea picowell array is in fluid communication with an inlet reservoirthrough a capillary inlet and with a waste reservoir through a capillaryoutlet. Liquid such as a first medium is added in the inlet reservoirand drawn by capillary action to and past the picowell array and to thewaste reservoir, optionally without needing pumps or complex interfaces.When the inlet reservoir is empty, a second medium is added to the inletreservoir and drawn by capillary action to displace the first medium toand past the picowell array and to the waste reservoir. A deviceincluding a picowell array in contact with flow is shown in the PCTpatent application published as WO2006/080000. In an exemplaryembodiment of the invention, a waste reservoir is provided sealed on allsides except for an inlet from the well array and including an air holeto release air. Optionally, a piece of absorbent paper or other suitablematerial is provided to wick fluid out of the reservoir.

In an exemplary embodiment of the invention, the capillary channelsserve to define a flow rate, so that even if medium is supplied at toohigh a rate, the actual rate reaching the cells is a known controlledrate.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details of construction and the arrangement of thecomponents and/or methods set forth in the following description and/orillustrated in the drawings and/or the Examples, if any. The inventionis capable of other embodiments or of being practiced or carried out invarious ways.

In addition it is noted that while the description focuses on picowellarrays, the manufacturing methods and capillary flow methods and/orother features described herein may be used with a cell holding area ofa different type, for example, an adhesive cell holding area. In anexemplary embodiment of the invention, a chemical coating or treatmentis provided on the surface of the substrate where the cells are about tosediment, e.g. Avidin-Biotin, poly-L-Lysine or Cell-Tack™ cell adhesionagents.

An embodiment of a device of the present invention, device 10, isdepicted disassembled in FIG. 1A.

In an exemplary embodiment of the invention, device 10 consistsessentially of five discrete components: a base plate 12, two spacers 14a and 14 b, an inlet reservoir component 16 and a cover 18. As shown,the components are provided in three planar layers, a base includingpicowells, a flow conduit defining layer and a cover. In an exemplaryembodiment of the invention, the cover defines capillary flow channelstogether with the flow conduit layer. Also, while the conduit defininglayer is shown to be made of multiple components, in some embodiments,it is provided as a single planar component, for example, a stack ofadhesive layers as described below.

In an exemplary embodiment of the invention, when provided as a singlelayer or as components, parts may be formed, for example, by diecutting, knife cutting, laser cutting, sand cutting and/or other cuttingmethod known in the art. Optionally, a single sheet of material orlaminate is cut using a die or laser to define multiple conduits and/orapertures, as needed, and then attached onto a large base or arrangementof base parts (e.g., optionally defining multiple devices in one or twodimensions). Optionally, the large base is cut after assembly intosmaller parts as needed. Optionally or alternatively, the layers arescored to make cutting and/or separation of individual components,easier.

Base plate 12 of device 10 is substantially a transparent glass (orplastic) microscope slide (e.g., 0.17 mm-1 mm thick, 2.54 cm wide, 7.62cm long, though other standard sizes may be supported) on which acircular 1 cm diameter well array 20 for the study of cells has beenembossed, for example from a UV-curable adhesive such as NOA-61, NOA-63or NOA-81 (Norland Products Inc., Cranbury, N.J., USA, USA). Embossingis performed, for example, by applying a drop of the fluid precursor ofthe adhesive and curing the fluid precursor while in contact with a die(made, for example, from a metal such as Teflon-coated metal, glass,PDMS or silicone rubber) having a negative of the well array. The dropof adhesive disperses between the slide and the die and forms a thinlayer (e.g., 10-100 micrometers thick). After the adhesive has set, thedie is peeled away or otherwise removed.

Optionally or alternatively to embossing, the glass is etched. In anexemplary embodiment of the invention, a layer (not shown) is providedover the glass, but not over the picowells, or not over the activepicowells, to match the height of the wells to the conduits. Optionallyor alternatively, the thickness of the conduit layers is selected tohave desired properties (e.g., of flow rate and/or cell dislocation),taking into account the thickness of the picowell area. In an exemplaryembodiment of the invention, the entire glass slide is etched orembossed with picowells, and these are covered over by adhesive tape oradhesive or another layer, except in area of the active picowells.

In an exemplary embodiment of the invention, the final thickness of thedevice is selected to be the same as a standard thickness of a glassslide. Optionally, the slide is provided with boundaries on one or moreside and the layers and components are placed within such boundaries.Optionally, the slide is pre-manufactured to include a recessed regionwhere the picowells and/or components and/or their cover are placed, sothat the slide is surrounded on one or more sides by original slidematerial. Optionally, such a structure provided a more robust device.

Optionally, additional step(s) are carried out to stabilize thestructure and its adhesion to the base and/or to extract and removenon-polymerized residues of the adhesive which may harm cells. Thesesteps optionally include one or both of annealing in mild temperature(˜60° C.) for at least one hour and then soaking in 90% ethanol for 5days. This process is optionally shortened if UV curing is longer and/orleave less monomers. Such soaking may be important to extract toxicun-cured agents from the polymerized adhesive. In a final step theslides are optionally rinsed in distilled water or fresh ethanol to washaway debris and/or residues of foreign materials.

FIG. 1B is a reproduction of a photomicrograph of the surface ofwell-array 20. It is seen that well array 20 is an array 20 micrometerdiameter 8 micrometer deep circular wells 22 hexagonally-packed that arejuxtaposed so that two adjacent wells 22 are separated by a wall 24 thatis less than 1 micrometer wide so that the distance between the twoadjacent wells 22 is not more than 110% of the well-dimensions (lessthan 21 micrometer interwell distance/20 micrometer well width). In anexemplary embodiment of the invention, flat bottomed picowells aremanufactured by DRIE (deep reactive ion etching) etching.

Spacer 14 may be made of one contour or created from several parts, suchas Spacers 14 a and 14 b and inlet reservoir component 16. The spacersare optionally about 0.5 mm thick and made of plurality of double-sidedadhesive sheets, with or without a strengthening layer in-between. Asdiscussed herein, the spacer height is one of the parameters which canbe modified to control the capillary forces and the fluid velocity. Anadditional such parameter is the materials used for spacer 14 and/or incontact with fluid. As will be discussed below, when device 10 isassembled, spacers 14 a and 14 b and inlet reservoir component 16define, at least partially, an inlet reservoir 26, an optional flowregulator 28 a, and a waste reservoir 30.

Cover 18 is optionally a thin (0.17 mm) glass cover slip or anothertransparent material, such as a flexible transparent plastic sheet.Optionally, the cover is preassembled with the device (such as beingpartly-adhered), prior to provision to an end user and/or prior to use.

Optionally, for assembly, the components are stacked together so thatthe adhesive of spacers 14 a and 14 b and inlet reservoir component 16adheres to base plate 12 and cover 18. Optionally, a separate adhesivelayer is provided, for example, being brushed or sprayed on.

Device 10 is depicted assembled in a perspective view in FIG. 2 and inside view in FIG. 3. The assembly of the various components helps defineinlet reservoir 26, flow regulator 28 a and/or waste reservoir 30.

An exemplary use of device 10 in implementing a method for studying aplurality of cells as individuals is described with reference to FIGS.4A-4F.

In FIG. 4A, a pipette 36 is used to apply a drop of a first liquid 38(e.g., a suspension of living cells having a diameter appropriate topicowells size, range e.g. 10 um to 400 um) into inlet reservoir 26defined by inlet reservoir component 16 and the edge of cover 18.Optionally, the distance between cover 18 and base plate 12 is 0.5 mm asdefined by spacers 14 a and 14 b. Such a distance is sufficient to drawfirst liquid 38 including cells suspended therein, by capillary action,into proximity of well array 20, through flow regulator 28 a and intowaste reservoir 30. As the space between base plate 12 and cover 18 isopen, air displaced by the introduction of the liquid escapes.

As depicted in FIGS. 4B and 4C, as first liquid 38 passes well array 20,cells 40 settle into individual wells 22. Flow regulator 28 a isrelatively narrow (2 mm wide) and so acts as a bottleneck, regulatingthe flow of liquids by slowing the flow of liquid such as first liquid38 therethrough. The flow-regulating effect substantially ensures thatcells 40 have time to settle in wells 22 and are not simply carriedaway. Cells 40 that do not settle in a well 22 are eventually washedaway by the flow of first liquid 38 to waste reservoir 30.

Optionally, after inlet reservoir 26 is empty of first liquid 38 (orwhile containing some liquid), a second liquid 42 is added to inletreservoir 26, as depicted in FIGS. 4D and 4E. Second liquid 42 is drawnby capillary action into and past well array 20, through optional flowregulator 28 a and into waste reservoir 30, all the while optionallydisplacing first liquid 38. In an exemplary embodiment of the invention,the flow-regulating effect of flow regulator 28 a reduces flow velocityso that cells 40 held in wells 22 are not displaced out of wells 22 andcarried away to waste reservoir 30 or move to other wells 22, therebybecoming unidentifiable. In an exemplary embodiment of the invention,the function of flow regulation is carried out by design of a capillaryinput to the picowells, capillary output form the picowells and/or addedbaffles in the flow.

Optionally or alternatively, cell dislocation is reduced by controllinga depth of a step from the flow to the picowells, which step may be zero(if wells on same level as inlet flow), negative (if wells above inletflow) or positive (if wells below inlet flow. Similarly, a step orincline between the picowells and outlet flow into the waste reservoirmay be adjusted accordingly. In an exemplary embodiment of theinvention, the start of the picowells and/or end of the picowells isspaced from such a step.

In an exemplary embodiment of the invention, such adjustments are madeduring manufacture and a user selects desired flow properties byselecting a certain slide design.

As depicted in FIG. 4F, when desired cells held in well array 20 may bestudied, for example by optical observation, with the help of amicroscope such as inverted microscope 44. In an exemplary embodiment ofthe invention, the cells are studies with a non-inverted microscope.

Flow Regulator

In an exemplary embodiment of the invention, a flow regulator, such asflow regulator 28 a, is a structure or feature that regulates the flowof liquids such as aqueous liquids past a well array such as well array20 in order to allow cells to more easily settle in a well array and toprevent cells from being displaced from wells in which held when liquidsare added.

In an exemplary embodiment of the invention, flow regulator 28 a ofdevice 10 is relatively narrow (2 mm across, 4 mm long) and therebyconfigured to act as a bottleneck to reduce the rate of flow of liquidsacross well array 20, so as to assist in filling wells 22 with cells 40and assisting in retaining cells 40 in wells 22 when liquids are addedto inlet reservoir 26.

Other embodiments of devices have other configurations of flow regulatorconfigured to reduce the rate of flow of liquids past a well-array.

In an exemplary embodiment of the invention, the flow regulator operatesby controlling the rate of capillary flow therein. Optionally, theseconsiderations also apply to designs where flow into the picowells isalso capillary, for example, as in FIG. 6 below. In an exemplaryembodiment of the invention, the rate of flow in the inlet capillary issmaller than in the outlet capillary or larger, as a means of affectingflow rate. Optionally, the capillary force applied by the reservoir isalso part of the control of capillary flow rate.

In an exemplary embodiment of the invention, the degree of retainingcells in their picowells during fluid manipulations (e.g., applied forstaining, treating with drugs and/or washing) is affected by thestructure geometry and the capillary flow characteristics, whereas theflow itself is determined by the conduit geometry and surfaceparameters.

In an exemplary embodiment of the invention, the flow rate through theflow regulator depends on two variables: the capillary cross section andthe angle of wetting of the fluid that flows when confronted with thematerials of the flow regulator, in addition to fluid properties, suchas viscosity, surface energy and/or specific gravity of cells in fluidrelative to rest of fluid. In an exemplary embodiment of the invention,the following estimating formula is used:F=Hσ _(LG1) cos θ₁+2Wσ _(LG2) cos θ₂ +Hσ _(LG3) cos θ₃

-   -   Where    -   H=height (clearance), W=Width. The indexes denote the different        surfaces: #1, 2, 3, which refer to top, sides and bottom which        may be made of different materials (e.g., or coated with such)        hence possessing different surface tensions and contact angle.        Optionally, different sides and/or top and/or bottom are coated        and/or made of different materials. This may cause the capillary        flow to have an asymmetric leading edge, which may affect cell        dislocations.    -   σ_(LG)=liquid-gas surface tension    -   θ=contact angle

The capillary pressure P is calculated by dividing the force by theapplicable area, HW, hence:P=(1/W)σ_(LG1) cos θ₁+(2/H)σ_(LG2) cos θ₂+(1/W)σ_(LG3) cos θ₃

In one example, if a same conduit was made from two glass surfaces theresulting flow velocity is too high due to the excellent wettability ofthe glass. If, however, the same flow conduit is made of Teflon, thehydrophobicity of Teflon could result in there being no capillary flowat all.

In an exemplary embodiment of the invention, a user can temporarilymodify the flow rate by changing the properties of the injected fluid.In an exemplary embodiment of the invention, some fluids, for examplefor staining and/or washing may be manipulated to have contact anglesand/or viscosities and/or otherwise have a different flow rate thatmatches the desired flow rates in the device, for example, by addingserum, glycerine or glucose.

In an exemplary embodiment of the invention, the slide to be used isselected base don the properties of the fluid and cells to be used andthe desired flow rate.

In FIG. 5A is depicted device 10 where flow regulator 28 a is relativelynarrow (e.g., depending on fluid, height and/or desired flow rate), thatis 2 mm across.

In FIG. 5B is depicted a device where a flow regulator 28 b is longer (8mm) and narrower (0.5 mm) so as to have a greater reduction of flow ratethan flow regulator 28 a.

In FIG. 5C is depicted a device where a flow regulator 28 c is narrow(0.5 mm) and serpentine (path length of 16 mm) so as to have a greaterreduction of flow rate than either flow regulator 28 a or 28 b.

In FIG. 5D is depicted a device where a flow regulator 28 d is 2 mmacross and 1 mm long, but is coated with a very thin layer of ahydrophobic material. The hydrophobic material reduces the rate of flowof aqueous liquids through flow regulator 28 d.

Optionally or alternatively, the regulator is higher than the rest ofthe flow conduit, thereby narrowing a cross-section of flow. Optionallyor alternatively, such added height may keep the main flow higher thanthe cell layer, possibly reducing cell dislocations. Optionally, cellsare placed in locations not expected to be affected by eddies in theflow caused by the change in height. Optionally or alternatively, thecells are close enough so that some flow-induced mixing between the flowand fluids near the cells, is supported.

Optionally or alternatively, a plurality of baffles, for example,obstruction are placed in the flow conduit. Optionally or alternatively,to baffles, other obstacles, such as traverse (to flow) scratches, areprovided, which scratches may cause slowing-down eddies and/or reducecapillary force.

Optionally or alternatively, a plurality of flow conduits are provided,for inlet and/or outlet form the picowells. Optionally, a user canselect which inlet conduit to use for a given pipettation, for example,based on expected flow rate characteristics of the added fluid in thatconduit.

In FIG. 5E is depicted a device where a flow regulator 28 e is as wideas well array 20 but includes edges 32 (at the interface between spacers14 a and 14 b with base plate 12 coated with a very thin layer of ahydrophobic material. The hydrophobic material reduces the rate of flowof aqueous liquids through flow regulator 28 e. At the same time, thewidth of flow regulator 28 e allows a more homogenous unidirectionalflow across well array 20, with relatively fewer eddies or stagnationpoints.

In FIG. 5F is depicted a device where a flow regulator 28 f is as wideas well array 20 but the entire width of flow regulator 28 f is coatedwith a very thin layer of hydrophobic material as in FIG. 5D. In FIG.5F, a relatively large region of base plate 12 is coated with a verythin layer of (relatively) hydrophobic material (e.g., a UV-curableadhesive such as NOA-61 or NOA-81) where an upstream portion 46 isembossed with a well array 20 while a downstream portion 48 is leftsmooth.

In FIG. 5G is depicted a device related to the device depicted in FIG.5F. The device is fashioned by placing a thin (20-100 micron) layer of a(relatively) hydrophobic material (e.g., a UV-curable adhesive such asNOA-61 or NOA-81) on substantially the entire surface of a base plate.Then, spacers 14 a and 14 b as well as inlet reservoir component 16 areplaced on top of the layer of hydrophobic material and a die including anegative of a well array is also appropriately placed. When thehydrophobic material is cured, not only is well array 20 produced aroundthe die, but spacers 14 a and 14 b as well as inlet reservoir component16 are fixed (by adhesion) to the base plate and all of flow regulator28 g and waste reservoir 30 include a layer of hydrophobic material.

In some embodiments, a combination of two or more of the above elementsis provided in one or more flow regulators (e.g., inlet and/or outlet).

Picowell Socket

In some embodiments of the invention, and as noted above, the picowellsmay be on a level lower than (e.g., be in a socket) or higher than(e.g., be on a pedestal) the flow conduits which lead to and/or from thewells. In some designs, the size and shape of such a difference in leveland/or distance of an edge of such pedestal or socket from the picowellscan have an effect on one or more of initial cell deposition and cellrepositioning during later flow. In many applications it is desirablethat the cells settle slowly after an initial application and that thecells not move during later flow. Optionally, the flow parametersdiscussed above are selected so the initial flow will be fast enough tocarry all cells into the picowell area. Optionally or alternatively, itis desired that at least by diffusion later flow will exchange foodstuff, stimulants, assay materials and/or waste products with the fluidin the picowells. Optionally or alternatively, it is desired that laterflow will wash any cells not in wells away from the wells.

In an exemplary embodiment of the invention, when the picowell is in asocket, a flow conduit is wider (e.g. is 4 mm wide) than the socket(e.g. diameter is 3 mm) and the extra width is selected to have desiredflow effects. Other relative widths that may be provided are 0.1 mmwider, 0.5 mm wider, 0.9 mm wider, 1.2 mm wider and 2 mm wider, orintermediate or greater widths. The socket and/or cell arrays may liesymmetrically or asymmetrically in the flow conduit.

As cells typically adhere to the wells with some force, there can be a“sweet spot” within which flow is fast enough but not too fast. In anexemplary embodiment of the invention, one or more of the following arevaried and/or taken into account:

(i) socket dimensions;

(ii) socket diameter (if round) vs. conduit width;

(iii) specific gravity of cells with regards to specific gravity of thefluid; and

(iv) picowells depth and wall geometry.

It should be noted that in some embodiments of the invention, thepicowells are mounted in the socket using a layer of double sided tapeunderlying a picowell chip.

Exemplary Design Process

In an exemplary embodiment of the invention, at least some of the aboveconsiderations are too complex and/or expensive to calculate or simulateand instead empirical design is used. In some cases, software is usedfor modeling and/or simulating a whole device or a part thereof, forexample, CFD-microfluidics software such as flow-3D by flow science inc,USA.

In an exemplary embodiment of the invention, the following process iscarried out:

(a) select some or all of picowells geometry, number of cells to beexplored (e.g., giving total number of picowells and cell chamber area)and/or cell sample volume (e.g., giving cell chamber height).

(b) select initial materials/coating. Materials involved in thestructure must first be proven as biologically inert (e.g., not toaffect the cell's life cycle) and optionally express low fluorescence(so as not to bias fluorescence measurements).

(c) modify sizes, materials and/or fluids to be used (e.g., using searchtechniques known in the art), until desired results are achieved. In anexemplary embodiment of the invention, such results are published in abook or database (e.g., stored on a computer readable media and/oravailable by network connection and/or via a computer with a CPU and amemory with instructions for an interface to the database) which can beconsulted as needed to select a proper set of slide, cells and fluidproperties and/or effects of incorrect fluid properties.

In some cases, a stable performance is not easy to achieve since thedevice must perform under various fluids types which are common on thebiology lab—water, buffers (e.g. PBS), wide range of cell growth media(with and without Serum, etc.), etc.

In an exemplary embodiment of the invention, the design process takesinto account other issues as well, for example, void volume and/oreddies caused when adding fluid. Typically, during the operation of thedevice fluids are added using a pipette. When the new drop of fluidtouches the fluid already in the fluid conduit, the fluids suddenlyconnect and a sudden “violent” fluid movement occurs. If this happen tooclose to the cells—cells might be shocked and dislodged. If, however thecontact point between the fluids is far from the picowells, this canresult in a larger dead volume from the pipettation point until a dropof reagent reaches and affect the cells. This can be a problem withrespect to one or both of time and final concentration of the reagentreaching the cells. Optionally, multiple pipettation points areprovided. Optionally or alternatively, a baffle is provided to absorbsuch violent movements, for example, in the form of one or moreobstructions or transverse scratches in the inlet flow.

In an exemplary embodiment of the invention, when adding fluid, morefluid than fits in the picowell socket is provided, for example, 8-9-10microliter for a 7 microliter socket. This can ensure overflow and/oravoid voids. In an exemplary embodiment of the invention, for example asshown in FIG. 6, overflow material which cannot flow fast enough intothe device can flow back via a channel that reaches away form thepicowells and pipettation location and towards an edge of the device,where it may be stored in a reservoir or drip out.

Base Plate

When a base plate is present, any suitable material or method may beused, optionally contingent on the above considerations.

In some embodiments, a base plate such as base plate 12 is an opticallytransparent material to allow observation of cells held in the wells. Bytransparent is especially meant transparent to one or more frequenciesof electromagnetic radiation in the visible, ultraviolet or infraredspectra.

Suitable materials from which a base plate is made include glasses(e.g., sodalime glass and borosilicate glass, especially optical gradeglass) and plastic materials (e.g., polyethylene terephthalate,polycarbonate, polyester, polystyrene).

In some embodiments of the invention, the substrate is solid and flat.In others, a hole may be formed therein (socket) for placing a picowellor other cell holding chip or section.

In some non-depicted embodiments, a base plate such as base plate 12 isthinner, for example is a 0.17 mm thick glass cover slip, allowinghigher resolution observation, because lens can be closer to the cells.Optionally, the cells are in a socket which is depressed and thereforethinner than the base plate.

Spacers and Inlet Reservoir Components

In exemplary embodiments of the invention, spacers and inlet reservoircomponents allow liquids to pass through device 10 by capillary action.In some embodiments, the thickness of the components is between 0.1 and1 mm. An inlet reservoir component such as 16 is the same height,thicker or thinner than corresponding spacers such as 14 a and 14 b,depending on the embodiment. Spacers and inlet reservoir components maybe fashioned according to any suitable method with which one skilled inthe art is familiar. For example, in some embodiments, spacers and/orinlet reservoir components are discrete components secured (for exampleby adhesion) to a base plate (as depicted for device 10). In someembodiments, spacers and/or inlet components are molded (e.g.,integrally formed with a corresponding base plate) or embossed in alayer of material, for example, from a UV curable adhesive (e.g., NOA-61or NOA-81) in a manner similar to the manner in which well-array 20 isfashioned as described above.

In some embodiments, the opening of inlet reservoir which is open to theenvironment is at least partially covered, for example so as to beexposed only through a narrow slit or hole to reduce evaporation. Insome embodiments, the opening (e.g., narrow slit or hole) is such toallow introduction of liquids (e.g., is of a size allowing entry of apipette tip).

Cover Slip

In some embodiments, a cover such as cover 18 functions as a part of theinlet reservoir, flow regulator and waste reservoir of a device, helpingto define these components as capillary channels, allowing the device tofunction without the need for external flow generators such as pumps orsyringes. In some embodiments, a cover is transparent, allowingobservation of cells held in a well array therethrough and/or allowingthe passage of light therethrough to illuminate the well array. Althoughcover 18 discussed above is of glass, other suitable materials may beused. In some embodiments, plastics or polymers (e.g., polycarbonate)are used and are optionally processed to desired shapes and/or contours.

Exemplary Well-Arrays

Exemplary well-arrays such as well-array 20 are generally as describedin the PCT patent application of the Inventor referenced above. In someembodiments the array of wells is disposed on a surface and comprises aplurality of wells having a dimension on the surface of no more thanabout 500 micrometers, no more than about 400 micrometers and even nomore than about 200 micrometers, the wells of a size and shape suitablefor retaining at least one cell therein.

Well-arrays may be fashioned using any suitable method from any suitablematerial, for example as described above or in any of the referenced PCTpatent applications of the Inventor.

In some embodiments, the wells of a well-bearing component are round orhexagonal, although in some embodiments, wells are triangular, square,pentagonal or any other suitable shape. Although wells of a well arrayof a well-bearing component may be arranged in any suitable arrangement,in some embodiments wells are hexagonally packed, allowing a highloading of cells per unit area.

In some embodiments, two adjacent wells of a well-bearing component arejuxtaposed and separated by a well-wall, for example as disclosed in thecited PCT patent applications of the Inventor. In some embodiments, thedistance between two adjacent wells is not more than 150%, not more than130%, not more than 120%, not more than 110% and even not more than 105%of the well-dimension.

The wells of a well-bearing component are optimally of any size so as tohold at least one cell or cell spheroid of a certain type. In someembodiments that are directed to the study of cells as individuals, itis generally preferred that the wells be small so as to avoid having alarge number of cells held in any one well.

For example, in some embodiments, a cell well-bearing component isconfigured for study of lymphocytes having a typically diameter of about6 micrometers. In some such embodiments a well-bearing component haswells of about 6 to 10 micrometer dimension on the surface of thewell-bearing component so that the lymphocytes enter and are held in thewells, one in each well.

For example, in some embodiments, a cell well-bearing component isconfigured for study of oocytes or single-cell spheroids having atypically diameter of about 400 micrometers. In some such embodiments awell-bearing component has wells of about 400 to 500 micrometersdimension on the surface of the well-bearing component so that theoocytes or spheroids enter or grow/assemble and are held in the wells,one in each well.

Typically, the dimensions of the wells are generally less than about500, 400, 200, 100, 50, 25 or even less than about 10 micrometers. Bydimensions is meant the usual meaning of the word and is dependent onthe shape of the wells. For example, for hexagonal or circular wells,the term dimension refers to diameter. For square or triangular wells ismeant the longest dimension of the square or triangle, respectively. Theexact size of wells of any given well-bearing component is determined bythe type of cells or alternately or additionally by the amount of cellsto be studied using the carrier. Since different types of cells havedifferent sizes, generally a carrier of the present invention will havewells of a size to accommodate one or more cells of the type to bestudied. Most preferred is that a well be of a size so as to hold nomore than one cell of the type to be studied at any one time. In otherembodiments, a well size is determined by the size of a predeterminednumber of a certain type of cells, so as to allow holding of thatpredetermined number of that type of cell, for example two cells, threecells, or even four cells.

To ensure that no more than a limited number of cells are held in agiven picowell, and that a held cell is able to efficiently absorbnutrients and release waste, the depth of a picowell is optionally notmore than the order of the size of the cell (or cell spheroid) which thewell is configured to hold.

In some embodiments, the wells have a depth no more than about 150%, nomore than about 120% and even no more than about 100% of the dimensionof the well on the surface. According to some embodiments, the depth ofthe wells is less than the dimension of the well on the surface. In someembodiments, the depth of a well is no more than about 500, not morethan about 200, not more than about 100, not more than about 50, notmore than about 30, not more than about 20 and even not more than about10 micrometers deep.

In some embodiments of the present invention, wells are dimples,depressions, or pits on the surface of well-bearing component. In otherembodiments, the wells are substantially enclosures of dimensions suchthat substantially an entire cell of a certain type is containablewithin the enclosure, each enclosure having an opening at the surface,the opening defined by a first cross section of a size allowing passageof a cell of the certain type.

In some embodiments, in or near a well array of smaller wells is locateda significantly larger well, serving as a collection enclosure. Selectedcells held in a specific well of the well array are moved, for examplewith the help of laser tweezers or the like, to a collection enclosure.

In some embodiments, a single device includes wells of different sizes,interspersed or in distinct regions whether in a single well array or ontwo or more well arrays. For example, in some embodiments one deviceincludes wells having 2, 3, 4 or even more distinct sizes. For example,in an embodiment, a single device includes wells having a diameter of 15micrometers, 20 micrometers, 100 micrometers as well as 250 micrometers.In some embodiments, such a combination allows the user to studyindividual cells as wells as clusters of cells on a single device. Insome embodiments, such a combination allows the user to purchase adevice configured to study cells of different sizes and the user studiesa region having a well array including wells configured to hold cellshaving the size of the cells of interest. Optionally, the sizes and/ordistance or other geometry of the picowells define areas where cellswill be located as individuals and where cells may conglomerate,multiply and/or communicate with nearby picowells.

Assembly and Packaging

In an exemplary embodiment of the invention, assembly and/or packagingis under vacuum, this can prevent and/or reduce formation of air bubblesduring storage and/or use. Optionally or alternatively, the surfaces tobe adhered are optionally sprayed with distilled water.

Such gas bubbles may be formed by gasses dissolved in the raw materials.Optionally, the above described annealing and/or washing are undervacuum, to remove gasses.

Exemplary Alternative Slide Design

Various devices useful for microscopy can be built based on an embossedmicroscope slide, for example a slide 0.17-1 mm thick. The glass is thencovered with a spacing layer which has a hole just above the embossedarea. The diameter of the hole may be smaller than the embossed area.This structure creates an open chamber, which can be useful foraccommodating a cell suspension. This structure may be covered by acover slip and cells can be observed by an upright and/or invertedmicroscope.

In another example, a slide structure supports one or more of exchangingcells media, staining, and/or other manipulations of the cells withintheir picowells. In such an embodiment, an additional “flow” layer iscured to form a flow channel above the cells chamber and a reservoir.This flow layer is sticky on both surfaces, and can optionally be bondedto the spacer layer beneath it, and to the cover layer above.Optionally, a flexible transparent cover, such as made of polycarbonatefilm, is bonded only on the distal edge of the flow channel. Optionally,a pinhole (or larger hole) is provided, for example, to drain air whenliquids are added during the work with the device. A non-stickyremovable liner is optionally provided keeps the cover from bonding tothe rest of the flow channel. In an exemplary embodiment of theinvention, the channels are bordered on either side, rather than open.

An exemplary such device 600 is shown in FIGS. 6A and 6B, with FIG. 6Abeing a blow-apart view. A glass slide 602 has embossed (or otherwiseattached) thereon a picowell array 604. A spacer layer 606 with anaperture 608 is attached above, for example, layer 606 being a two sidedadhesive layer. A flow layer 610 including a flow channel 612 and areservoir 614 is provided above, optionally as a two sided adhesivelayer. A cover layer 616 with an optional air hole 624 and slot 618 areprovided above. Optionally, a non-adhesive layer 620 is provided betweenlayers 616 and 610 and includes a tip 622 which is temporarily attachedto layer 610.

Optionally, as shown in FIG. 6B, the length of layer 620 is shorter thanlayers 616 and 610, so layers 616 and 610 can be adhered to each otherat their tip.

Other structures may also be built up by layering shaped layers ofdouble-sided adhesive, on microscope slides and/or other bases.

In use, flexible covers 620 and 616 are pulled back and expose thechamber formed by aperture 618 and wells area 604. Cells are loaded intothe chamber and removeable liner layer 620 is peeled off. Flexible cover616 is released and bonds to the rest of upper surface of flow layer616. Layer 610 optionally defines a capillary channel between layers 616and 606. Aliquots of fluid can be presented to the capillary slit 618created between the cover and the opening of the flow channel. Capillaryforce pulls the fluid into the channel, the fluid replaces and/or flowsover the cell media, and the excessive fluid is gradually accumulated inthe reservoir 614. Additional fluid can be presented to the flowchannel, basically until the reservoir is full. Optionally, fluid can beremoved from reservoir 614

Double sided adhesive tapes, optionally medical grade, or other adhesiveagents, may be used to bond the layers and/or to form the layers.

If double sided tapes are used, the layers are optionally bonded whilewet. This can eliminate air and prevent such air from being trappingbetween the layers, possibly minimizing the appearance of air bubbles inthe suspension along incubation time. Optionally, the devices aremanufactured and/or packed under vacuum, to prevent repeated airpenetration between the layers before the device is used.

FIGS. 6C-6H are engineering drawings showing exemplary sizes of featuresof device 600.

FIG. 6C is a top view of device 600, showing layer 620 folded back overthe other layers.

FIG. 6D is a top view of a slide layer 602, optionally formed of glass,including active picowell area 604 which may have picowells engraved,embossed and/or adhered thereto.

Exemplary materials for spacers (e.g., between layers of double sidedtape) include:

-   -   By Rogers corp. CT, USA: Silicon rubber or polyurethane rubber,        e.g. MC-300, MC-600, MS-6000, MS-SOFT, HT-200, HT-1200, HT-1500,        HT-6135, HT-6200, HT-6360 series, MS series    -   By Du-Pont-Kapton®, Mylar®, Neoprene.    -   By Silex, Hampshire, UK: Silicone rubber sheeting grades GP40,        50, 60 & 70    -   By Kleerdex LLC (PA, USA): Kydex®    -   By GE plastics (see below): Lexan

Exemplary materials for double sided tapes include:

-   -   By 3M, MA, USA: adhesive tapes #1504, 1509, 1510, 1513, 1517,        1522, 1524, 1524A, 1577, 1772, 1773, 1774, 9500, 9731, 9776,        9874, 9877, 9887, 9889, 9917, 9960, 9971

Exemplary materials for the cover include:

-   -   By General Electric plastics (MA, USA): Lexan (8010, 8020, 8030,        8040, 8A13, 8A23, 8A35, 8A37, 8B25, 8B26, 8B35, 8B36, FR25,        FR60, FR63, FR65, FR66, FR700, FR83),    -   By SKC inc, GA, USA: Skyrol (SH71, SH81, SH82) and other        Polyester (PET, PET-G etc.) films.

In an exemplary embodiment of the invention, the combination actuallyused depends on desired flow rates and actual geometries. Optionally, aset of materials and geometries is chosen to have a set of propertiessuitable/acceptable for a plurality of situations, even if not optimalfor all.

FIG. 6E is a top view of a base layer 606 including an aperture 608which optionally expose sonly some picowells to the rest of the deviceand/or which serves to attach a picowell chip in place. Layer 606 isoptionally formed of spacer material with double sided tape laminatedthereon, above. In an exemplary embodiment of the invention, layer 606has a thickness of between 0.2 mm and 2 m, for example, 1 mm.

FIG. 6F shows a conduit layer 610, with exemplary dimensions forreservoir (e.g., 300 micro liters) and inlet conduit. Optionally, thislayer is formed of a sandwich (e.g., by laminating) of double sidedtape, 175 micron thick spacer and a third layer of 1522 above.Optionally, trapped air is avoided using a distilled water spray and allresidues are removed.

FIG. 6G shows a temporary sticking avoiding layer 620, with a handle 623and a fold point 621. Optionally, this allows the layer to be foldedover, adhered to underlying layers only at a side far from where a coverlayer is attached and then easily removed. Optionally, the layer is madeof HDPE coated with silicone at points where it is not to stick to 1522layer below.

FIG. 6H shows a cover layer 616, with air hole 624. This layer isoptionally formed of 0.175 mm thickness.

In an exemplary embodiment of the invention, device 600 is configured sothat an initial flow (first 10 micro liters) is about 48 seconds forcell growth media (RPMI 1640). And 30 seconds for Buffer (PBS).

In an exemplary embodiment of the invention, the average flow rate ofthe succeeding aliquots is about 0.5 μl/sec in media and about 0.75ul/sec in PBS. In an experiment using molt4 cells (e.g., which appear tobe more prone to dislodging), this resulted in total cell movement(after repeating adding 6 aliquots of 10 ul) of up to 10% in RPMI cellmedia and up to 2% PBS buffer.

In an exemplary embodiment of the invention, device 600 has an activearea and working volume of 3.2 sq. mm. (Approximately 18,000 wells of 20microns), containing 7.5 micro liters of cells suspension. The lengthwidth and/or thickness are of a standard microscope slide. Optionally,the total device volume is 300 micro liters. Optionally, there are nomoving parts in use and the device is in one piece when unpackaged,possibly simplifying packaging.

FIG. 7 illustrates an alternative slide design 700, in which a chip orother element 703 with a picowell area 705 formed thereon (part or all)is mounted on a base layer 702. In an exemplary embodiment of theinvention, a layer of double sided tape 707 having an aperture formedtherein acts as a mask to expose only some of the picowells to the restof the device and/or to attach chip 703 to layer 702. Optionally, asshown a cover 716, reservoir 7614 and capillary conduit 712 are providedas above.

Exemplary Usage Process

FIG. 8 is a flowchart of a method of using device 600, in accordancewith an exemplary embodiment of the invention.

At 802 a device is selected form a set of devices, according to desiredcell holding characteristics and/or flow characteristics, taking in toaccount, for example, cell size, cell adhesion, fluids used and/or fluidcontact angle with materials in device/

At 804, cover 616 is pulled back.

At 806, spacer layer 620 is removed. As noted above, spacer 1620 mayfolded and at the side opposite of where cover 616 is adhered to device600, include a handle 623 and a portion adhered to layer 610.

At 808, cover 616 is prevented from falling down on layer 610, byinserting a wedge at the point where the previously adhered section oflayer 616 meets the previously unadhered portion thereof. Optionally,the wedge is a small cylinder having a length of the slide width and adiameter of 2 mm to 5 mm, optionally made of a material, e.g., Teflon ora silicone which is not prone to adhere to the adhesive layer. Othermeans of preventing the fall of cover 616 can be used as well.

At 810, the device can be laid down on a flat surface, leaving bothhands free.

At 812, cells are added to the exposed aperture 1608, optionally with nocapillary movement. This may also preload the device, cell area and/orflow inlets with fluid.

At 814, the wedge is removed.

At 816, cover 616 is adhered to layer 610, optionally with care that noair bubbles are formed, optionally, with wetting of cover 616beforehand. Optionally, this is carried out by one or more of manualpressure or a roller or a hand held stamp.

At 818, or before cover closure, the cells are allowed to settle inwells.

At 820, the cells are imaged and/or otherwise tested. Optionally, suchimaging includes adding various reagents and/or stimulants, into slot618 (822). In some cases, some time is waited so such added fluids havea desired effect (824). Acts 820-824 may be repeated several timesand/or for a period of time, for example, minutes, hours, days or weeks.

Optionally, at 826, the cover is removed, so the cells can be retrievedand further tested/processed (828). In an exemplary embodiment of theinvention, cover 616 includes a tear line above conduit 612, which istorn manually. Optionally or alternatively, a knife is used to cut cover616. Optionally, a jig is provided which positions a cutting edgerelative to a standard microscope slide, for such cutting. Optionally oralternatively, the adhesive layer of the top conduit is made of anon-permanent adhesive, which can be separated and reattached.

Embossing

In an exemplary embodiment of the invention, a cell holder is formed byembossing. In an exemplary embodiment of the invention, a base iscovered with an optionally liquid substrate, contacted with a die andthe substrate is at least partially hardened. Optionally, the diedefines a plurality of pico liter wells in the substrate. Optionally,the substrate is adhesive to the base, but not to the die, optionally inspite of the substrate being smooth and the die having small features.Optionally, the die and adhesive and base materials are selected toafford such differential adhesion. Optionally, the die is removed beforethe substrate hardens. In an exemplary embodiment of the invention, atleast two areas on the substrate are patterned, each with a differentfinal pattern of cell holding regions.

In an exemplary embodiment of the invention, one or more walls orbarriers are added to the cell holder before, during or after embossing.

In an exemplary embodiment of the invention, the base is notmechanically prepared for the substrate.

In an exemplary embodiment of the invention, there is provided a holderdevice having at least one cavity for receiving a sample of cells in amedium, wherein the at least one cavity includes a substrate havingrefractive index substantially equal to that of water, includes amultiplicity of pico liter wells (also called “picowells”, herein)formed therein and has a generally inert wall.

In an exemplary embodiment of the invention, there is provided a methodof making a holding device having a substrate characterized by an indexof refraction essentially equal to that of water, which is adhered to acarrier plate, wherein the substrate is formed into a multiplicity ofpico liter wells.

In an exemplary embodiment of the invention, there is provided a methodof making a holding device having at least one cavity for receiving amedium, wherein the at least one cavity has a multiplicity of pico literwells formed in a substrate having a refractive index substantiallyequal to the medium in which the cells are carried.

In an exemplary embodiment of the invention, there is provided a methodof making a die for forming an array of picowell arrays. Optionally, thedie is assembled from individual well forming die elements. Optionally,a handle with a plurality of clamps to hold the die elements is used. Inother embodiments, other means to hold die elements together into atemplate are used. Optionally, the die also serves as a template fornon-well embossed elements.

In an exemplary embodiment of the invention, there are provided cellholding devices including a plurality of picowell arrays, on each one,optionally in the form of a macroscopic array of picowell arrays.Optionally, the picowell arrays are separated by barriers which may haveselected blocking ability, for example, being fluid blocking or cellblocking or fluid passing.

A holding device for studying cells in a medium in accordance with anexemplary embodiment of this invention comprises at least one cavitydefined by generally inert wall surrounding a substrate. The substratehas a surface in which a plurality of pico liter wells is formed. Thesubstrate is translucent and has a refractive index substantially equalto the refractive index of the medium.

An exemplary holding device for studying cells in accordance with anexemplary embodiment of this invention comprises a substantiallytransparent substrate having a refractive index of 1.33. The substratehas a multiplicity of pico liter wells formed in an upper surface and awall structure attached thereto.

In another embodiment, an exemplary embodiment of the present inventionincludes a holding device for studying cells comprising a substantiallytransparent carrier plate having a plurality of cavities surrounded bywalls formed in a first surface of the carrier plate, a layer ofadhesive, which is known to those skilled in the art, disposed on abottom surface of each cavity, a layer of substantially transparentsubstrate material having a refractive index of 1.33 and having amultiplicity of pico liter wells formed in an upper surface thereofdisposed on the adhesive layer. While MY-133 is known to have arefractive index close to that of water, heretofore MY-133 hasapparently not been considered suitable for use as an inert substratefor holding live cells in a medium. In other embodiments, other adhesivematerials, possibly with other refractive indexes are used forembossing.

In an exemplary embodiment of the invention, the embossing methodsdescribed herein are applied on fragile bases so little temperatureand/or pressure changes are applied. Rather the embossing is optionallyat low pressure and at room temperature (e.g., 15-35 degrees Celsius).In an exemplary embodiment of the invention, the embossed substrate isnon-reversibly deformed by the embossing. In an exemplary embodiment ofthe invention, the embossed substrate is set by one or more of chemicalsetting, light setting, radiation based setting and/or othercross-linking or hardening or setting methods known in the art and asappropriate for the specific materials used.

In an exemplary embodiment of the invention, the thickness of theembossed layer is minimal, for example, being 15, 5%, 20%, 60%, 100%,300%, 100% or intermediate percentages of the depth of the picowelland/or other embossed feature. Optionally or alternatively, theadditional thickness below the well is 1, 10, 20, 30, 100 microns orintermediate amounts. Variations of the embossing methods describedherein, which may be variously referred to as “stamping”,“micro-stamping”, “replicating” or “soft-lithography” may also bepracticed in accordance with some embodiments of the invention.

In an exemplary embodiment of the invention, embossing can includemodification of the picowells in addition to changing the geometry. Forexample, the template can have attached thereto patterns for the wallsbetween the picowells, medicaments and chemicals for attachment to thepicowells or walls between them, small beads for such transfer and/orelectrode wires. In portions that are not for picowells, electronics,valves and/or other items may be impressed on the substrate duringembossing.

In an exemplary embodiment of the invention, embossing is carried ourusing a rolling die (e.g., a cylinder). Optionally, the glass slides arecut after embossing. Optionally, unneeded picowells are masked by alayer with an aperture defining the active picowells.

While the above description has focused on forming picowells, otherstructures can be formed as well in like manner, instead of picowellsand/or to support picowell operation. For example, such structures caninclude landmarks to assist in user orientation (e.g., patternedclusters of non-etched areas or other singularities or markings toenhance user's orientation when the array is used). Optionally, somesuch markings are particularly visible under a microscope (e.g., picowell address and/or a well address). In another example, micro-fluidicsstructures, such as reservoirs, channels, mixers, filters and flowregulators are embossed (e.g., with the embossing defining 3D structuresthat affect flow. Optionally or alternatively, preparation for variousstructures is embossed (and/or additional elements mounted on the die),f or example, pumps, electronics, conductors, sensor mountings and/orvalves.

The embossing process may change in accordance with the specificmaterial or precursor used for the substrate. For example, somematerials require the application of a primer layer to strengthen thebonding to the base layer, whereas, additionally or independently, othermaterials require a detoxification process in order to extractbiologically toxic residues such as un-cured monomers in the embossedlayer.

For example, a typical precursor may be a UV-curable adhesive such asNOA-61 or NOA-81 or the low self-fluorescence NOA-63 (Norland ProductsInc., Cranbury, N.J., USA, USA). Embossing is performed, for example, byapplying a drop of the fluid precursor of the adhesive on glass orplastic base, and curing the fluid precursor while in contact with a die(made, for example, from a metal such as nickel, glass, PDMS or siliconerubber) having a negative geometry of the well array. The drop ofadhesive disperses between the slide and the die and forms a thin layer(e.g., 10-100 micrometers thick). After the adhesive has set, the die ispeeled away or otherwise removed.

Additional acts are optionally made to stabilize the structure and itsadhesion to the base and/or to extract and/or remove non-polymerizedresidues of the adhesive which may harm cells. Such acts can includeannealing at a mild temperature (˜60° C.) for at least one hour.Optionally or alternatively, the bases are soaked in 90% ethanol (orother suitable solvent, depending on materials used for construction)for 5 days. This can extract toxic un-cured agents from the polymerizedadhesive. Optionally or alternatively, the slides are rinsed indistilled water to wash away debris and/or residues of the extractedundesired foreign materials. Optionally or alternatively, the materialschosen and/or washing are selected to allow long term life of biologicalcells without toxic effects form the cell holder. Optionally, the cellsreside 1 hour, 10 hours, 24 hours, 2 days, 4 days, 1 week, 1 month ormore in the cell holder without such toxic effects.

Exemplary base plates for any of the embossing methods described hereininclude any substantially transparent glass (or plastic) surface, suchas a microscope slide (e.g., 0.17 mm-1 mm thick, 2.54 cm wide, 7.62 cmlong), a Petri dish (either monolithic plastic dish or a plastic dishwith a thin glass or polymer sheet bottom, designed for high resolutionmicroscopy) or the bottom of microtitter plates (either monolithicplastic plate or plate having a thin glass or polymer sheet bottom,designed for high resolution microscopy).

Embossing can take place on essentially whole bottom area, or in certainareas. The size of the embossed area is determined by the applied volumeof the precursor and the actual size of the die.

In some embodiments, the Petri dish or any other pico liter wellsbearing device may include a partitioning element, such as a plastic orfabric barrier, to distinguish between several areas in the same device.The partitioning element, single or plural, if made or include mesh,slits or perforated parts can also act as a flow regulator, to damp andcontrol (by selecting proper size and pitch of the openings) fluidtransfer between partitions. For example, such a barrier can block orreduce fluid flow or block or reduce cellular or particle flow (e.g., ofcertain sizes), for example, acting as a filter. In some embodiments,the barrier serves to reduce flow rates to levels which would not removecells/particles from picowells. Such barriers may be attached by anymethod known in the art. Optionally, the barriers are attached bydipping in a UV curable adhesive as used herein. Optionally, suchbarriers include a base ring for mechanically aligning said barriers onsaid substrate.

In this and other embodiments, the coverage of the carrier by picowelland/or other embossed regions can be, for example, 1%, 5%, 10%, 20%,50%, 70%, 90%, 100% or intermediate amounts, for example, depending onthe application.

In an exemplary embodiment of the invention, multiple sizes and/or typesof pico liter wells are provided on a same substrate, for example, 2, 4,6, 10, 20 or intermediate numbers of types of pico wells may beprovided.

In those cases where plate consists of an upper structure and a bondedbottom, a precursor is optionally uniformly applied to the transparentbottom prior to placing the die and irradiating. Optionally, pico literwells are created on the whole bottom area (e.g., using a flat anduniform die). The bottom plate is then bonded to the upper structure asit is usually done in this type of plates (for example, based onutilizing NOA as a cementable adhesive). Optionally or alternatively,pico liter wells may be created only at desired locations, such as thoseareas which will become the bottom of wells, excluding inter-wellsareas. This can be controlled, for example, by applying aliquots ofprecursor instead of a uniform layer.

Embossing, using MY-133 or NOA or materials may be practiced on amicroscope slide, either on the whole glass or selected parts and mayserve as a base plate for various single or multi cavity devices. Forexample, the embossed glass can be covered with adhesive spacers andcovers already used in laboratory practice, such as those made by GraceBiolabs (Bend, Oreg., USA)www.gracebio.com/Products/Imaging_Microscopy/CoverWell_Perfusion_Chambersand enhance the practice to include pico liter wells bottoms instead ofa plain glass surface. For example, these covers can form a water-tight,multiwell cell incubation or cytochemistry chamber when pressed tocoverslips or microscope slides. In some embodiments, reagents can bequickly added and removed through access ports without disturbing orcross-contaminating specimens in adjacent wells, while cells remain intheir picowells.

Exemplary Non-Slide Devices

FIGS. 9A and 9B illustrate non-slide devices using masking and/or doublesided table to build up walls, in accordance with exemplary embodimentsof the invention. In an exemplary embodiment of the invention, suchdouble sided tape serves to bond a wall onto a substrate and/or fill inunused picowells so fluid does not flow therein.

FIG. 9A shows a multi-well device 900, optionally of the standard sizeof a microtitter plate (e.g., ˜127 mm×85 mm), with for example, 24, 48or 96 wells, constructed using a base layer 902 having picowells formedover substantially its entire surface. On base 902, a double sidedadhesive layer 904 is provided, with apertures 905 matching desired wellareas. An upper layer 906 defines the well walls for a plurality ofwells 907.

FIG. 9B shows a Petri dish 920 formed using a base layer 922 withpicowells defined thereon. A double sided adhesive layer 924 is used todefine active picowells and to mount a wall layer 926 above. Optionally,as shown, layer 924 includes an aperture 928 for accessing somepicowells and also includes a cover portion 930 which mounts on layer924 and also prevents cells from adhering to layer 924. Optionally, theouter diameter of adhesive layer 924 is smaller than an outer diameterof cover portion 930. If larger, excess is optionally trimmed, forexample, with a knife.

Multi-Channel Slide

In an exemplary embodiment of the invention, a slide is provided withmultiple picowell areas. While such areas may be provided in series(each one down flow of the other), in other embodiments, such picowellareas are provided to have parallel fluid pathways. Optionally, thedesign of FIG. 6 is made narrower and/or smaller and two or more suchdesigns provided on a single slide. Optionally or alternatively,multiple such fluid pathways can share an input inlet and/or wastereservoir (but not the cell area itself or capillaries leading to andfrom), so two or more sets of cells can receive same treatments.Optionally or alternatively, one or more input inlets lead to two cellholding areas, while at least one input inlet is provided for only oneof the cell areas, allowing both same and different treatment ofmultiple cell areas.

FIG. 10A shows a multi-picowell design in which the waster reservoirs(or alternatively, input reservoirs) are implemented as elongatedconvoluted channels, rather than as wide areas. IN a different devicedesign, such a waste channel may be defined as a spiraling channelsurrounding or extending form a pico cell area.

As shown, a base layer 1002 has two picowell areas 1004 and 1004′defined thereon, for example, by embossing, engraving and/or adhering. Amasking and/or filling and/or matching layer 1006 has defined thereinaperture 1008 and 1008′ which define which picowells will be active. Anext, conduit, layer 1010 defines two waste reservoir channels 1015 and1015′ which end at covered sections 1014 and 1014′ including a fixedcover 1017 with air holes 1024 and 1024, for air release. As notedabove, the size of air holes can be used to affect capillary flow rate.

A cover 1016 (for example flexible, e.g. polycarbonate, or rigid e.g.glass) may operate as cover 616 above and a pair of fluid adding ports1018 and 1018′ may be coupled to picowells by capillary or non-capillaryflow.

FIG. 10B shows an alternative embodiment with two side by side cellholders and conduits, in which the design of FIG. 6, for example, isnarrowed, so two will fit n a standard slide, side by side. In anexemplary embodiment of the invention, the reservoir is made narrower,but the capillaries are not, so as to maintain flow rates. Optionally oralternatively, multiple design changes are made, for example, narrowingcapillaries while using more wettable materials in the capillaries, tomaintain desired flow characteristics.

In an exemplary embodiment of the invention, different ones of thecapillaries of different fluid systems on a same base have differentflow rates. Optionally or alternatively, different ones of the cellholders are adapted for holding different cells and/or have differentcell position disruption rates.

In an exemplary embodiment of the invention, the reservoirs and/orinlets associated with the two cell holders are shared and/or linked,allowing, for example, to provide same treatments to two sets of cellsand/or provide a larger reservoir.

FIG. 10C shows an eight section cell holding device, with eight side byside cell holders and conduits. Optionally, the device is of a size of astandard microtitter plate. Other numbers of cells holders and conduitsmay be provided, for example, 16, as shown in FIG. 10D, 48, 96 or largeror intermediate numbers.

Optionally, two or more reservoirs, or all the reservoirs are linked toform a unitary waste receptacle. Optionally, this receptacle is largeenough so that danger if flow back into cell holders is small.Optionally, the widening of the capillary from the cell holder to thereservoir, serves as a one way flow path, wherein flow back is lesslikely.

In an exemplary embodiment of the invention, the systems of FIG. 10C andFIG. 10D (and optionally of 10B) are separable into individual slide orgroups of slides. Optionally, this is achieved by scoring the upperlayers and/or base plate. Optionally, such devices (as in FIGS. 10A-10D)are formed by methods other than the layering methods described herein.Optionally or alternatively, the base layer is pre-cut.

In an exemplary embodiment of the invention, the inlet locations of theindividual cell holders are positioned uniformly in a geometrycompatible with standard multi-well cell holders. This can allowautomatic fluid providing to such cell holders. Optionally, initialloading is manual. Optionally, the cover layer is separated for eachcell holder, so that each cell holder can be uncovered, filled andcovered (e.g., adhered in place) separately. Optionally, separatenon-stick layers sections are provided for each cell holder. In analternative embodiment, cell filling is into an inlet, and is alsoperformed automatically. This may be facilitated by a design such asthat of device 10.

Potential Advantages and/or Features

Potential advantages and/or features of device 10 and 600 and otherembodiments described herein, which may be utilized in accordance withexemplary embodiments of the invention, include one or more of thefollowing. It is noted that some of the advantages and/or features canbe provide din other device designs in accordance with exemplaryembodiments of the invention, for example the shapes and flow controlcan be achieved in devices not formed of layers as in FIG. 6. Inaddition, not all features and/or advantages are provided in everydevice in accordance with exemplary embodiments of the invention.

(a) Devices that are relatively simple to manufacture and assemble atlow cost and in high volumes, for example, by providing simple partsoptionally cut from sheets to assemble with optionally reduced need foralignment and/or processing of details on a part or assembled parts.

(b) Devices which support high-resolution imaging by both upright andinverted microscopes.

(c) Devices over a range of desirable flow rates, cell sizes, array sizeand/or other properties, which are optionally made from modules, whichare interchangeable and/or which are for this reason and/or othersamenable for construction on a single assembly line.

(d) Devices which are formed only of thin planar layers, optionally withlimited breakability, with capillary channels defined by cutting out oflayers, rather than cutting away into layers. Optionally, the thicknessof the channel is controlled by varying the number of sub layers thatlaminate to form a conduit layer.

(e) Devices which do not require assembly of discrete parts by a user,but which are optionally substantially protected from air and/or fluidsexcept at very limited portion thereof. Optionally, the devices allowadditional manipulation prior to sealing of such a device by a user.

(f) Devices which mount walls on a microfluidics device using doublesided tape.

(g) Using a layer of double sided tape to mask out a larger cell holdingarray, for example a pico well array, to define a part of the array tobe active.

(h) Controlling capillary flow by a combination of one or more ofcapillary cross-sectional shape and/or size, capillary materials, airrelease, baffles and/or surface treatments.

(i) Ability to select a design which has desired flow and/or celldislodging characteristics, for a particular need and/or experimentalsetup.

(j) Devices with capillary channels including reservoirs which aresealed on all sides except for an inlet and air release or absorbingmedia.

(k) Preventing contamination and/or reusability by adhering a cover ontop of a device, while optionally distancing a cell holding area from aninlet port so such area is reachable only by capillary flows and cannotbe washed out. Adhesive can be, for example, permanent or multi-use(enabling repeated opening and closing).

(l) Setting a cover on a device using an adhesive attachment between thecover and the conduits, so that the cover defines the conduits,initiates and/or supports capillary action and is optionally unbreakable(unlike thin glass covers).

(m) Provide sets of devices (e.g., 2, 3, 4, 5, 10, 15, 20, or more orintermediate numbers or an array of different devices connectedtogether) as a kit for a range of experimental settings (e.g., desiredflow rate) conditions, cells and/or reagents and/or provide devicespackaged with instructions or at least an indication of expectedperformance under different conditions.

(n) Provide devices with flow regulation at both inlet and outlet fromcell holding area.

(o) Allow overflow to leave device without compromising the cell holdingarea.

(p) Provide devices which have unique identification mark or productcode—barcode or RFID—to enable automatic upload of devicecharacteristics.

(q) By including an opening to the atmosphere near the cell holder,diffusion of gasses in and out of the cells are supported. Optionally,such diffusion is reduced or prevented by taping or otherwise placing acover over the inlet. Optionally, the distance between the inlet portand the cell holders is selected to have a desired gas diffusion rate.

In an exemplary embodiment of the invention, sides are marked with amarking or instructions which indicate various properties thereof, forexample, flow rate under one or more conditions, cell/well size, celldisruptability, size and/or picowell arrangement and/or diversity.

In an exemplary embodiment of the invention, there is provided anexperimental design software which includes a table indicating variouscharacteristics of such devices and which can be used to select devicesthat have desired flow, etc. characteristics for certain experiments.Such software can be provided, for example, on computer readable mediasuch as a CD, diskette, hard-disk, ROM and/or RAM. Optionally, themarkings include a computer readable marking such as a barcode or RFID.

In an exemplary embodiment of the invention, software (e.g., local or ata network location/server) is used by a user to order particular devicedesigns and such devices are assembled according to the request, frommodular components.

Optionally, the parts of the device are sold separately, optionally insets of various properties and a user or manufacturer can assemble suchdevices, as needed.

While the description has focused on manual manipulation, a plurality ofslides can be managed by computerized/robotic means. Optionally, the useof a capillary to control flow rates, simplifies handling of such slidesand allows nutrients, etc. to be provided as a drop at an inlet port,which drop will then be “pumped” at a desired rate. This allows rapidtreatment of multiple wells and cell holding areas without individuallycontrolling the flow at each of a plurality of cell arrays usingindividual pumps or in serial manner.

General

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of means “including and limited to”.

The measurements described herein for, inter alia, sizes, volumes and/orrates, while exemplary, may be modified depending on the application,and are intended to include, for example, measurements that are forexample, 10%, 20%, 30%, 50%, 70%, 90% larger or smaller, or 100%, 200%,300% larger, or intermediate percentages of the above percentages.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

The word “exemplary” is used herein to mean “serving as an example,instance or illustration”. Any embodiment described as “exemplary” isnot necessarily to be construed as preferred or advantageous over otherembodiments and/or to exclude the incorporation of features from otherembodiments.

The word “optionally” is used herein to mean “is provided in someembodiments and not provided in other embodiments”. Any particularembodiment of the invention may include a plurality of “optional”features unless such features conflict.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. In caseof conflict, the patent specification, including definitions, willcontrol.

In addition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

The invention claimed is:
 1. A cell study device, comprising: a baselayer wherein said base layer comprises a socket; a planar conduitdefining layer, separate from said base layer, including an apertureforming side walls of a channel therein; a planar cover layer, separatefrom said base and conduit defining layer, which, together with the sidewalls of said channel and said base layer, forms an enclosed capillaryflow channel in said conduit layer in parallel to said base layer andabove said base layer, wherein the cross-section of said capillary flowchannel is generally perpendicular to said base layer and is sized suchthat capillary fluid flow results from the attachment of said coverlayer to said conduit layer, wherein fluid flow in said capillary flowchannel is capillary fluid flow resulting solely from capillary forceswithin said capillary flow channel; and a cell holding area positionedwithin said socket comprising an array of cell-holding picowells on saidbase layer in fluid contact with said capillary flow channel, saidpicowells being sized and configured to accommodate and immobilize asingle cell or a predetermined number of cells for exposure to asequence of liquid aliquots delivered by the capillary flow channel,thereby permitting study of cells individually, wherein said capillaryfluid flow in said capillary flow channel is horizontal and above saidpicowells and wherein said cover layer is movable to permit access tosaid cell holding area.
 2. A device according to claim 1, wherein saidcell-holding area is formed in said base layer.
 3. A device according toclaim 2, wherein said cell holding area includes at least oneorientation mark useful for identification under microscopy.
 4. A deviceaccording to claim 2, wherein a part of said cell holding area is maskedby a masking layer underlying said conduit layer.
 5. A device accordingto claim 1, wherein said cell holding area includes a plurality ofcell-holding well arrays in fluid communication with said capillary flowchannel.
 6. A device according to claim 1, wherein said cover layerincludes one or more air holes in communication with said cell holdingarea for air release.
 7. A device according to claim 1, wherein saidcapillary flow channel terminates at a distal end thereof in asubstantially sealed waste reservoir with no fluid exit.
 8. A deviceaccording to claim 1, wherein said capillary flow channel terminates ata distal end thereof in a substantially sealed waste reservoir with anabsorbent material as a fluid exit.
 9. A device according to claim 1,wherein said cover layer includes a fluid inlet area for said capillaryflow channel.
 10. A device according to claim 1, packaged in vacuum. 11.A device according to claim 1, wherein said conduit layer is permanentlyadhered to said cover layer.
 12. A device according to claim 1, whereinsaid device is provided with a removable non-stick layer between saidcover layer and said conduit layer, removal of said non-stick layerpermits capillary flow in the channel between said cover layer and theconduit layer.
 13. A device according to claim 1, wherein said conduitlayer is temporarily adhered to said cover layer.
 14. A device accordingto claim 1, wherein said conduit layer includes an adhesive on both itsfaces.
 15. A device according to claim 1, wherein said layers areselected of dissimilar materials with dissimilar contact angles withfluids.
 16. A device according to claim 1, wherein said device has theform factor of a standard microscope slide.
 17. A device according toclaim 1, wherein said device has the form factor of a standardmicrotiter plate.
 18. A device according to claim 1, wherein said layersare formed of materials that do not interfere with cell behavior for atleast 24 hours when loaded with an aqueous solution.
 19. A deviceaccording to claim 1, further comprising indications of one or both of acapillary flow rate and a cell dislocation rate therein.
 20. A deviceaccording to claim 19, comprising a plurality of different cell studydevices, each with different sets of flow rate and dislocation rate. 21.A device according to claim 19, wherein said indication is per one orboth of fluid property and cell type.
 22. A device according to claim19, wherein said indication is machine-readable.
 23. A device accordingto claim 19, wherein said indication is human readable.
 24. A deviceaccording to claim 19, including instructions explaining saidindications.
 25. A device according to claim 19, including software on acomputer readable medium for using said indications.
 26. A deviceaccording to claim 1, further including a removable non-adhesive layerthat interferes with adhesion of at least two parts of said device, saidinterfering inactivating said capillary flow channel.
 27. A deviceaccording to claim 26, wherein said adhesion is permanent, when saidnon-adhesive interfering layer is removed.
 28. A cell study deviceaccording to claim 1 comprising: a double-sided adhesive layer maskingsome of the picowells in said cell holding area, wherein said conduitdefining layer is mounted on said double-sided adhesive layer.
 29. Acell study device according to claim 28, including a plurality of cellholding areas that are in fluidic isolation from each other.
 30. A cellstudy device according to claim 29 wherein each cell holding areaincludes a capillary flow channel separate from capillary flow channelsassociated with the other cell holding areas.
 31. A cell study deviceaccording to claim 28 wherein said adhesive layer is apertured.
 32. Acell study device according to claim 28, wherein said walls are at least2 mm high.
 33. A cell study device according to claim 1, wherein saidcover layer is transparent.
 34. A cell study device according to claim1, wherein said cover layer is flexible.
 35. A cell study deviceaccording to claim 1, wherein said cover layer is partially adherent tosaid conduit layer.
 36. A cell study device according to claim 1,wherein said cell-holding array is defined by a plurality of closelypacked pits in a surface of said base layer.
 37. A cell study deviceaccording to claim 1, wherein said channel walls are at least 2 mm high.38. A cell study device according to claim 1, wherein said cell-holdingwell array is comprised of picowells having a diameter of about 1micrometer or about 500 micrometers.
 39. A cell study device accordingto claim 1, wherein said base layer is comprised in a structure havingthe form of a Petri dish.
 40. A device according to claim 1, wherein thearray of cell-holding picowells is located in a recess in said baselayer.
 41. A cell study device according to claim 1, wherein thepicowells comprise non-adhesive picowells configured to preventadherence of cells and the cell-holding area geometry is configured tocontrol cell dislocation rate.
 42. A cell study device according toclaim 1, further including a flow regulator to prevent cells from beingdisplaced from the picowells due to fluid flow.
 43. A device accordingto claim 1, wherein said picowells are formed of hydrogel.
 44. A cellstudy device comprising: a unitary structure including a base layer,wherein said base layer comprises a socket; the base layer including acell holding area positioned within said socket, wherein said cellholding area comprising an array of picowells in which the picowells aresized and configured to accommodate and immobilize a single cell or apredetermined number of cells; a second layer permanently adhered to thebase layer including an aperture that defines a barrier on the baselayer and provides fluid access to the picowell array; and a furtherlayer forming an upwardly extending wall permanently attached to thesecond layer surrounding the picowell array; the device furtherincluding a cover that is separate from or part of the unitarystructure, which together with said unitary structure forms an enclosedcapillary flow channel in parallel to said base layer, wherein thecross-section of said capillary flow channel is generally perpendicularto said base layer and is sized such that capillary fluid flow in saidcapillary flow channel is-in parallel to said base layer, and above andin parallel to said picowells results from the attachment of said coverlayer to said unitary structure, and wherein said cover is moveable toallow access to said picowell array.
 45. A cell study device accordingto claim 44, wherein the base layer includes a structure that surroundsthe cell holding area.
 46. A cell study device according to claim 45 inwhich the structure forms a recess in the base layer in which thepicowell array is located.
 47. A cell study device according to claim44, in which the cell holding area is comprised of picowells having adiameter of about 1 micrometer or of about 500 micrometers.
 48. A cellstudy device according to claim 44, in which the unitary structure is inthe form of a Petri dish.
 49. A cell study device according to claim 44,in which the cover and the base layer are transparent.
 50. A cell studydevice according to claim 44, in which the array of picowells isembossed on the base layer.
 51. A cell study device according to claim44, in which the cell holding area includes at least one orientationmark useful for identification under microscopy.
 52. A cell study deviceaccording to claim 44, including at least one partitioning elementproviding fluidic isolation between areas of picowells on the baselayer.
 53. A cell study device according to claim 44, in which thepicowell array is defined by a plurality of closely packed pits etchedin a surface of the base layer.
 54. A cell study device according toclaim 44, in which the cell holding area is coated with a chemical orbiological material.
 55. A cell study device according to claim 44, inwhich the base layer is glass.
 56. A cell study device according toclaim 55, in which the second layer, the wall surrounding the secondlayer, and the cover are formed of a plastic material.
 57. A cell studydevice according to claim 44, in which the cover is moveable and isconfigured to fit on the top of the upwardly extending wall.
 58. A cellstudy device according to claim 44, in which the picowell array iscomprised in a component attached to the base layer as part of theunitary structure.
 59. A cell study device according to claim 44,wherein the picowells comprise non-adhesive picowells configured toprevent adherence of cells and the cell holding area geometry isconfigured to control cell dislocation rate.
 60. A cell study deviceaccording to claim 44, further including a flow regulator to preventcells from being displaced from the picowells due to fluid flow.
 61. Acell study device according to claim 44, wherein said picowells areformed of hydrogel.